The bacterial ribosome has become one of the better understood cellular organelles through the application of a variety of structural techniques; a large part of the primary structure of both protein and nucleic acid components have been determined. Despite this abundance of information the molecular mechanism of ribosome function remains illusive. It is necessary to extend the refine further the structural studies. This proposal deals with determining the protein topography of the ribosomal subunits using crosslinking between neighboring proteins. The reagent 2-iminothiolane will continue to be used to form sulfhydryl derivatives of protein amino groups in intact ribosomal subunits. Mild oxidation causes the formation of disulfide bonds some of which are intermolecular links between neighboring proteins. Diagonal (polyacrylamide/dodecyl) gel electrophoresis is used to analyze the crosslinked proteins. The pattern of crosslinks is reproducible and characteristic. The analysis of all crosslinks in each subunit will be continued to the complete analysis of the diagonals. At this time analysis of the 30S subunit is 80% complete, that of the 50S subunit 30%. The diagonal represents a fingerprint of proteins interactions under specified conditions. Effects of changing the extent and specificity of modification and of all conformation on crosslinking will be investigated. Changes will be identified by inspection of diagonal gels once all components there have been identified for standard conditions. Specific functional sites will be defined by crosslinking: the binding sites for initiation factors IF2 and IF3, the subunit interface region between 30S and 50S particles. There is convincing evidence that many proteins are elongated in the ribosome. The major long-range goal of this project is to refine crosslinking procedures to the point when information on the secific "contact regions" between pairs of proteins can be identified. This will involve peptide mapping to identify sites of modification and crosslinking. The refined information will greatly increase the accuracy of the map of ribosomal protein topography. This in turn should lead to new insights into the mechanism of protein synthesis..